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Abstract

Background—Premature ventricular contractions (PVCs) commonly coexist with cardiomyopathy. Recently, PVCs have been identified as a possible cause of cardiomyopathy. We developed a PVC-induced cardiomyopathy animal model using a novel premature pacing algorithm to assess timeframe and reversibility of this cardiomyopathy and examine the associated histopathologic abnormalities.

Methods and Results—Thirteen mongrel dogs were implanted with a specially programmed pacemaker capable of simulating ventricular extrasystoles. Animals were randomly assigned to either 12 weeks of bigeminal PVCs (n=7) or no PVCs (control, n=6). Continuous 24-hour Holter monitoring corroborated ventricular bigeminy in the PVC group (PVC, 49.8% versus control, <0.01%; P<0.0001). After 12 weeks, only the PVC group had cardiomyopathy, with a significant reduction in left ventricular ejection fraction (PVC, 39.7±5.4% versus control, 60.7±3.8%; P<0.0001) and an increase in left ventricular end-systolic dimension (PVC, 33.3±3.5 mm versus control, 23.7±3.6 mm; P<0.001). Ventricular effective refractory period showed a trend to prolong in the PVC group. PVC-induced cardiomyopathy was resolved within 2 to 4 weeks after discontinuation of PVCs. No inflammation, fibrosis, or changes in apoptosis and mitochondrial oxidative phosphorylation were observed with PVC-induced cardiomyopathy.

Introduction

Premature ventricular contractions (PVCs) are a very common entity associated with cardiomyopathy (CM) and other cardiac diseases, and yet their effects on the cardiovascular system are not well understood. Although the presence of PVCs may carry adverse prognosis, especially in structural heart disease, PVCs in general are thought to be benign or secondary to the cardiomyopathic process. Recently, a few small studies have reported a relationship between high PVC burden and left ventricular (LV) systolic dysfunction.1–4 Moreover, other observational and nonrandomized studies demonstrated improvement of LV function after a successful PVC suppression strategy.1,4–8 These observations led to the description of an entity called PVC-induced CM.1–6 Consequently, a current clinical conundrum is to recognize when PVCs are responsible for the development of a CM or secondary to a CM.9

The cardiovascular effects of PVCs have not been prospectively or systematically studied primarily because of the lack of an animal model and the unpredictability and variability of PVCs in the clinical setting.10,11 We describe a novel PVC animal model with a unique pacemaker algorithm to demonstrate the link between frequent PVCs and LV systolic dysfunction.

Methods

Animal Preparation and Pacemaker Implantation

Under general anesthesia, 14 mongrel dogs (2 to 3 years old; weight, 35 to 45 lbs) underwent implantation of an experimental pacemaker (details are in the online-only Data Supplement). Through a left thoracotomy, a bipolar epicardial single-lead (Medtronic model 4968) was sutured in the right ventricular (RV) apex. Appropriate lead position was acceptable for R-wave sensing above 4 mV and pacing threshold <2 V at 0.5 ms. Experiments were approved by the McGuire VAMC Institutional Animal Care and Use Committee. Three weeks after surgical recovery, a baseline echocardiogram and 24-hour continuous 3-lead Holter monitoring were obtained before initiation of PVC protocol (outlined below). One animal was excluded from the cohort because of a baseline abnormal LV ejection fraction (EF).

Simulation of Frequent PVCs

A persistent high PVC burden originating from the RV apex was simulated through premature pacing algorithm (described below). Premature pacing algorithm was programmed to deliver PVCs in a bigeminal pattern (1:1 ratio or 50% PVC burden) with a fixed coupling interval of 250 ms (240 bpm) after each intrinsic ventricular sensed event (Figure 1). Pacing threshold was obtained during implantation and on a biweekly basis. Pacing voltage output was programmed at least twice the diastolic threshold to ensure ventricular capture.

The PVC coupling interval may be programmed as either fixed (programmable between 190 to 375 ms) or adaptive. The adaptive coupling interval is determined as a percentage of average cycle length of prior cardiac sensed events (Figure 2). In contrast to fixed coupling interval, adaptive coupling interval can only be used when premature pacing burden is programmed to <33% (as a minimum of 2 R-R intervals are needed to obtain an average cycle length of cardiac sensed events).

PVC Protocol

The dogs were randomly assigned to either PVC (n=7, enabled premature pacing algorithm) or control groups (n=6, disabled premature pacing algorithm). The control group had a device programmed for sensing only (ODO). After group assignment and initiation of PVC protocol, an echocardiogram was repeated at 2, 4, 8, and 12 weeks to follow changes in LV function in all dogs. Twenty-four-hour, continuous, 3-lead ECG Holter monitoring was repeated at the end of the 3-month protocol. After 3 months, 4 animals from the PVC group and 3 from the control group were euthanized, and cardiac tissue was excised and frozen for further analysis. The remaining 3 animals assigned to the PVC group were allowed to recover for 4 weeks (recovery phase) by disabling the PVC algorithm (online-only Data Supplement Figure 1).

Cardiac Evaluation

Two-Dimensional Echocardiography

Echocardiograms were performed at baseline, 2-weeks, and every 4 weeks throughout the duration of the PVC protocol, using a commercially available system (Sequoia c256 Siemens). Animals assigned to the recovery phase had additional echocardiograms at 2 and 4 weeks after discontinuation of PVCs. LVEF, fractional shortening (FS), end-systolic and end-diastolic LV and left atrial dimensions, LV thickness, LV compliance (E/A and E/E′ ratios), and the severity of mitral regurgitation were evaluated using standard criteria by the American Society of Echocardiography12,13 (online-only Data Supplement). Tissue Doppler imaging was used to assess the timing of local contractility (QRS-to-contraction) in 4 different LV locations in reference to the QRS complex (lateral base, septal base, mid lateral wall, and midseptum). LV dyssynchrony was assessed by the standard deviation of QRS-to contraction time between these locations.14 PVCs were suspended (algorithm disabled) at least 15 minutes before echocardiography to obtain an accurate calculation of described parameters. The echocardiographic measurements were performed offline by a cardiologist blinded to the randomization arm.

Ventricular Effective Refractory Period

Programmed ventricular stimulation (S1S2) with different drive trains (S1: 300 ms, 350 ms, and 400 ms) were performed to determine ventricular effective refractory period (VERP). VERP was defined as the longest S1S2 that did not cause myocardial capture.

Myocardial Microscopic Evaluation

Four different LV samples (2 from the LV apex and 2 from the LV anterior wall) in each canine (PVC group, n=4; control group, n=3; online-only Data Supplement Figure 1) were obtained and stained with hematoxylin and eosin and Masson trichrome to assess inflammation and fibrosis, respectively. Leukocytic infiltrates, grade of fibrosis (score 0 to 4+), and percentage of fibrosis were assessed in 10 random fields per sample (×10 and ×40 magnification, respectively). Two LV samples (LV apex and anterior wall) in each animal underwent the TUNEL (terminal deoxynucleotide transferase-mediated nick-end labeling assay) technique (DNA fragmentation, Oncor, Gaithersburg, MD) to assess apoptosis as previously described.15 Apoptotic nuclei were counted in 4 to 5 random fields per sample. The apoptotic index was expressed as the number of apoptotic cells of all cardiomyocytes per field16 (online-only Data Supplement).

Isolation and Analysis of Cardiac Mitochondria

Cardiac mitochondria were isolated and analyzed according to Palmer et al,17 with minor modifications as previously published.18,19

Oxygen consumption in subsarcolemmal mitochondria and interfibrillar mitochondria were measured using a Clark-type oxygen electrode at 30°C with glutamate (complex I substrate) and succinate+rotenone (complex II substrate).18 Further details are provided in the online-only Data Supplement.

Statistical Analysis

Sample size was calculated to reach statistical power of 80% with type I error of 0.05 for LV dysfunction after a 12-week period of ventricular bigeminy (details are in the online-only Data Supplement). All data are expressed in mean±SD. Statistical analysis was performed with the use of with SAS/STAT Software (SAS Institute, Inc, Cary, NC). A repeated-measures ANOVA was performed to compare temporal changes in LVEF between study and control groups. We calculated the change score (Δ) from baseline to 12 weeks for all echocardiographic and VERP data for each animal in the PVC and control groups. A 2-sample t test was used to compare the mean change score at 12 weeks between PVC and control groups. The degree of mitral regurgitation at 12 weeks was compared between PVC and control groups by use of the Mann-Whitney U test. A probability value <0.05 was considered significant.

Results

Premature Pacing Algorithm

Thirteen animals underwent device implantation without surgical complications. Canines were randomized to PVC (n=7) or control (n=6, without PVCs) groups. No device or algorithm malfunctions were noted during the study. Twenty-four-hour Holter monitoring showed ventricular bigeminy in the frequent PVC group with an average PVC burden of 49.8±0.01% compared with 0.01±0.001% in the control group (P<0.001). The PVC protocol increased the average heart rate from 81±7 to 130±13 bpm (P<0.001). Figure 3 illustrates ventricular bigeminy with a 2-lead ECG tracing and rate histogram in a single animal before and after premature pacing algorithm is enabled.

Cardiac Function

Echocardiographic findings are summarized in the Table. A new LV systolic dysfunction was observed in the PVC group after 12 weeks of ventricular bigeminy with a 34% relative reduction in LVEF (Figure 4), a 45% decrease in FS, and a 39% increase in LV end-systolic dimension (LVESD) (Table). In contrast, no changes in LVEF, FS, or LVESD were noted in the control group. LVEF and FS were significantly lower and LVESD was significantly greater in the PVC group after 12 weeks compared with the control group. Mitral regurgitation (semiquantitative, zero to 3+) reached significant difference between PVC and control groups (control, 0.58±0.2 versus PVC, 1.29±0.7; Mann-Whitney U test; P=0.04; Table). However, no animal had signs of overt heart failure such as lethargy, decreased activity, fluid retention, or tachypnea. Furthermore, no significant difference in LV end-diastolic dimension, LV wall thickness, left atrial size, or left atrial area was found after 3 months between PVC and control groups (Table).

LVEF in PVC (n=7) and control groups (n=6) during 3-month follow up. A 4-week recovery period in the noneuthanized PVC group canines (n=3) demonstrates normalization of LVEF (bars represent standard deviation).

Myocardial Microscopic Evaluation

Despite LV systolic dysfunction, the PVC group (n=4) did not demonstrate increased inflammation, degree of fibrosis (PVC, 1.75±0.5 versus control, 1.3±0.8), percentage of fibrosis (PVC, 5.4±1.7% versus control, 6.5±3.9%), or apoptotic index (PVC, 2.85±1.77 versus control, 2.59±0.64; online-only Data Supplement Figure 2) when compared with the control group (n=3). Inflammatory infiltrates were absent in both groups (online-only Data Supplement Figure 3). These findings are purely descriptive, and no statistical results were performed because of small sample size.

Oxidative Phosphorylation of Cardiac Mitochondria

Maximal rates of ADP-stimulated respiration and the coupling of respiration were unchanged in cardiac mitochondria in the PVC group compared with the control group (online-only Data Supplement Table 2).

Discussion

The cardiovascular effects of PVCs have not been systematically studied because of the absence of animal models. In the present study, we have developed a novel animal model, using a unique premature pacing algorithm to reproduce PVCs and corroborate the clinical entity of PVC-induced CM. The major findings of this model are (1) ventricular bigeminy induced CM characterized by a reduced LVEF and enlarged LV systolic dimension; (2) PVC-induced CM was reversible within 4 weeks after cessation of PVCs; and (3) PVC-induced CM lacks histopathologic abnormalities such as inflammation, fibrosis, increased apoptosis, or abnormal mitochondrial oxidative phosphorylation.

Canines were selected in our PVC model because of their similarity to humans of the cardiac His-Purkinje system20 and their extensive description in tachycardia-induced CM models.21 The premature pacing algorithm is capable of reproducing different PVC burdens (Figure 2). Ventricular bigeminy was chosen as a clinically significant PVC burden (50%) that would probably result in PVC-induced CM and demonstrate the concept that frequent PVCs alone can induce LV dysfunction in otherwise normal hearts. Because the heart rate of the dog ranges from 60 to 200 bpm, the pacing stimulus (PVC) was delivered at a fixed coupling interval of 240 ms (250 bpm) after ventricular sensed event to ensure bigeminal pacing even at faster rates of 200 bpm (300 ms).

The echocardiographic findings in our animal model are consistent with previous results of retrospective/observational clinical studies of PVC-induced CM.1,2,4–8 As our report was finalized, an animal model using 2 RV leads connected to a dual-chamber pacemaker reported similar echocardiographic findings after 4 weeks of ventricular bigeminy.22 In addition, our model demonstrated that (1) LV dysfunction developed as early as 2 weeks and continued to decline for the following 10 weeks (Figure 4) after initiation of ventricular bigeminy without clinical evidence of heart failure, (2) severity of mitral valve regurgitation increased, and (3) VERP was likely to prolong after chronic bigeminy, whereas (4) CM was reversible as demonstrated by the recovery of LV function and normalization of LV dimensions within 4 weeks after cessation of PVCs.

Importantly, we found a trend of VERP to prolong in this PVC-induced CM model. This was not surprising because VERP prolongation and “electric remodeling” has been previously reported in failing hearts.23 Electric remodeling has been characterized by alterations in intercellular ion channels, which result in prolongation of action potential duration, VERP, and slowing of conduction.23 Further investigations are needed to clarify and explain electrophysiological changes and how these relate to the proarrhythmic effects of frequent PVCs reported in patients with and without LV systolic dysfunction.24,25

The histopathologic and metabolic features of PVC-induced CM have never been described. Tissue analysis in this animal model did not show inflammation, fibrosis, or increased apoptosis after 3 months of high PVC burden. The unaltered respiration with complex I and complex II substrates suggests that mitochondrial electron transport was not significantly altered by exposure to PVCs despite the decrease in LV systolic function. On the basis of complete recovery of LV systolic function after cessation of PVCs, it is not surprising that there were no gross structural abnormalities. We believe that this CM is secondary to a functional rather than a structural abnormality due to the lack of gross structural abnormalities in our animal model. For instance, abnormalities in calcium handling could potentially translate in myocardial dysfunction.

The mechanism(s) by which PVCs induce CM are unknown. Two major theories have emerged: (1) a short PVC coupling interval6,26 and (2) LV dyssynchrony during PVCs. A short PVC coupling interval in subjects with high PVC burden would result in an overall increase in the average heart rate and “ tachycardia,” possibly leading to a pathophysiology similar to a tachycardia-induced CM. We believe that this animal model of CM is clearly distinct from tachycardia-induced CM because the average heart rate with PVCs (130±13 bpm) is significantly lower than described in a tachycardia-induced CM dog model (heart rate >180 bpm).14,21,27 In addition, the absence of fibrosis, increased apoptosis, and mitochondrial dysfunction as well as the normalization of LV diastolic dilatation after cessation of PVCs supports a distinct mechanism from tachycardia-induced CM.14,21,28–31 Alternatively, frequent PVCs may cause LV dyssynchrony similar to chronic RV pacing,32–35 which has been associated with higher mortality rates and a greater incidence of LV dysfunction.6,36 The abnormal pattern of electric activation and LV dyssynchrony34,37–39 resulting from these PVCs may cause disruption and further progression of dyssynergic LV wall motion.40–42 However, the time course to develop LV dysfunction in this model is quite different from the sole effects of long-term RV pacing.43,44 Finally, we propose the chronic effects of “postextrasystolic potentiation” as a third mechanism of PVC-induced CM. This phenomenon was studied extensively in the 1970s when coupled pacing was postulated to be beneficial for the treatment of heart failure. An increase in intracellular Ca2+concentration and myocardial oxygen consumption was demonstrated with postextrasystolic potentiation,45,46 which could also contribute to the development of CM.

To the best of our knowledge, our PVC canine model with chronic ventricular bigeminy describes for the first time the time course of echocardiographic findings, changes in VERP, and the histopathologic and mitochondrial characteristics of PVC-induced CM. Nevertheless, the minimum PVC burden required to induce CM remains unclear. Furthermore, it is uncertain if different sites of PVC origin and coupling intervals would affect the development and/or severity of PVC-induced CM. In contrast to the use of a dual-chamber pacemaker recently reported22 to reproduce ventricular bigeminy, our novel premature pacing algorithm is able to provide different PVC burdens (from 5% up to 75%) and coupling intervals that mimic different clinical scenarios (Figure 2).

Limitations

PVCs simulated through a pacemaker in our animal model are not intrinsic, but cardiac bipolar pacing represents local myocardial depolarization similar to a spontaneous ventricular event. RV apical pacing was performed, and it is unclear if these results may be extrapolated to PVCs from other cardiac sites. We cannot exclude that tachycardia plays a lesser role because the average heart rate was increased with premature pacing algorithm. However, this is similar to the clinical scenario investigated in patients with high-burden PVCs. Our histopathologic analysis was limited to anterior and apical segments of the LV. We cannot exclude the presence of regional abnormalities in the remaining LV walls. Similarly, we cannot exclude the possibility that extended exposure to frequent PVCs beyond 12 weeks could result in significant cardiac remodeling with chronic irreversible structural changes.

Novelty and Significance

This study validates the premise that frequent PVCs can result in a reversible LV dysfunction in structurally normal hearts. Even if PVCs are the result of CM, PVCs by themselves may contribute to and further worsen CM and heart failure symptoms.8,9 This findings support further clinical studies in patients with CM associated with frequent PVCs.

Most importantly, this novel premature pacing algorithm and PVC animal model will facilitate further scientific evaluation of the cardiovascular effects of PVCs in structurally normal hearts and other established heart failure models.

Conclusions

In summary, a novel premature pacing algorithm has allowed the study of the clinical entity of PVC-induced CM in structurally normal hearts. The PVC-induced CM canine model demonstrates that frequent PVCs with a bigeminal pattern alone can cause reversible LV dysfunction within 2 weeks, which appears to progress throughout the 3 months of continuous PVCs. Finally, PVC-induced CM lacks histopathologic and mitochondrial abnormalities seen in other canine models of CM.

Disclosures

Dr Huizar received grant support from St Jude Medical and was a clinical investigator for Biotronik. Dr Kaszala was a clinical investigator for Boston Scientific, St Jude Medical, and Sorin. Dr Ellenbogen received grants and honoraria and was a clinical investigator and consultant for Boston Scientific, Medtronic, St Jude Medical, and Biotronik. Dr Wood was a clinical investigator and speaker for Boston Scientific, Medtronic, and St Jude Medical.

Acknowledgments

We thank St Jude Medical for providing experimental devices. We thank Susan Quinn and Richard Klaty for their commitment to this project and animal care and Harsha Kannan (Virginia Commonwealth University) for technical help in the pathological laboratory.

Clinical Perspective

Premature ventricular contractions (PVCs) are a common entity associated with cardiomyopathy and other cardiac diseases, and yet their effects on the cardiovascular system are not well understood. This is primarily because of the lack of animal models and the unpredictability and variability of PVCs in the clinical setting. With the use of a novel premature pacing algorithm capable of reproducing different clinical scenarios of ventricular ectopy, the effects of chronic ventricular bigeminy in structurally normal hearts were studied in an animal model. Our canine model validates and describes for the first time the time course of echocardiographic findings, changes in ventricular effective refractory period, and the histopathologic and mitochondrial characteristics of PVC-induced cardiomyopathy. These findings support further clinical studies in patients with cardiomyopathy associated with frequent PVCs because the minimum PVC burden, origin, and coupling interval required to induce cardiomyopathy remains unclear. Finally, this novel premature pacing algorithm and PVC animal model will facilitate further scientific evaluation of the cardiovascular effects of PVCs in structurally normal hearts and other established heart failure models.